1. Field of the Invention
This application incorporates by reference in its entirety U.S. patent application Ser. No. 08/299,608 now U.S. Pat. No. 5,614,186, inventor Charles M. Rush.
This invention relates to the treatment of plants to reduce the plant's susceptibility to severe disease caused by virulent viral pathogens. Plants inoculated with a non-virulent or mildly virulent virus do not express severe disease symptoms caused by the virulent virus even when co-inoculated with the virulent virus. The novel inoculation method includes application of viruliferous fungus to seeds by coating seeds with survival structures of the viruliferous fungus, such as cystosori of Polymyxa species.
In addition, the present invention relates to a method for delivering viruses, both wildtype and recombinant, to plants. Soilborne fungi containing recombinant, and/or wildtype, viruses are used to infect plants and thereby deliver recombinant and/or wildtype viruses to plants. In an embodiment of the present invention survival structures, such as cystosori, of soilborne fungi containing recombinant or wildtype viruses are used to coat seeds of plants susceptible to soilborne fungi such that plants growing from these seeds become infected with the recombinant or wildtype virus containing fungi thereby delivering the virus to the plant.
In addition, the present invention relates to a method for delivering foreign genes or viruses to plant cells. Specifically, the present invention relates to a method of introducing foreign genes or viruses into plant cells in planta, via a seed treatment method utilizing recombinant furoviruses and their natural fungal vectors in order to introduce specific agronomic traits, and production of desirable products such as pharmaceuticals.
2. General Background
Applied biotechnology in agriculture has been moving at an astonishing pace since the successful transformation of tobacco via Agrobacterium (Ti plasmid) was reported in 1983 (32). Over the last ten years, transformation of major crop species with foreign genes in order to introduce desirable agronomic traits has been a primary research focus in the scientific community. Today, new varieties have been developed from all the major crop species in which transgenic plants have been produced (65) and many of them are now available to farmers and consumers (87). The traits that have been or soon will be incorporated into crops include herbicide tolerance (Roundup, Basta, Buctril, Atrazine) insect (Bacillus thuringiensis endotoxin) and disease (chitinase, virus coat protein) resistance, quality improvement (high-stearic-acid oilseed rape, pre-colored cotton, and high methionine maize seed), and high-value biopharmaceutical products (alkaloids and vaccines) (24,87).
The methods for delivery of a single gene or set of genes to plant cells are known as transformation and transfection. These methods have the goal of incorporation of the alien gene(s) into plant cells or integration into the plant genome. This permits the expression of the foreign genes transiently or permanently. Transient expression describes a result of attempted transformation in which expression of the gene (production of mRNA) is time-limited, usually to a period of a few days or weeks. Reduction of expression may or may not be accompanied by loss of the gene itself. In contrast, permanent expression is accompanied by the transmission of the expressing foreign gene to subsequent generations.
Transfection is a process in which viral genes are transferred into plant cells through normal or modified virus activity. The viral genome may be modified to allow transfer of non-viral genes that replicate episomally in the cytoplasm or integrate into the plant genome (91). Transformation describes any of several other methods, both biological and physical, for the transfer of genes which are stably integrated into the genome. Such experiments sometimes result in transient expression of the gene. These methods include, but are not limited to, tissue-culture-dependent DNA delivery systems, such as Ti-plasmid (72, 76), PEG (97), Ca.sup.2+ -mediated DNA uptake through protoplasts (70), liposome encapsidation, electroporation (65), microinjection (30), silicon carbide fibers (57), particle bombardment (59), as well as other plasmid-mediated gene transfers, and tissue-culture-independent systems, such as DNA injection into ovaries (23), DNA delivery along the pollen tube (69), and DNA uptake through seed imbibition (92, 105). A necessary feature of gene delivery systems is the establishment of marker genes (NPT II, CAT, BAR, HYG, GUS and LUC) (65), promoters (NOS, CaMV 35S, Ubi 1, Adh 1, Act 1 and pEMU) and introns which, in combination with the promoter regions, facilitate the selection of transgenic plants and the expression of the foreign genes (17). In general, for all the reproducible gene transfer procedures, there are biological limitations associated with their applications, such as host specificity, genotype specificity for tissue-culture-regeneration capacity, somaclonal variation, fertility loss, insufficient level of gene expression, instability of transgenes, and potential for transfer of foreign genes into weed species (65). Most processes are highly technical, expensive, lengthy and difficult to control. At present, for dicot crops, the most efficient and widely-applicable transformation method is the Agrobacterium vector system. For monocot crops, particle bombardment of shoot tips or immature embryos is the current method of choice, even though successful Agrobacterium-mediated transformation of maize and rice have been reported (49, 52). Both procedures limit the number of genotypes that can be manipulated, dependent on their tissue-culture-regeneration capacity. Below is a more detailed description of these currently available gene transfer methods along with their limitations.
Agrobacterium-Mediated Transformation
Agrobacterium gene delivery has become almost routine in many laboratories but transformation of some species using this technology has been unsuccessful. Agrobacterium causes crown gall disease in many dicot plants in nature by transferring a defined segment of DNA from its tumor-inducing (Ti) plasmid into the nuclear genome of cells through a wound area of the plants (58). This unique plant-microbial interaction has been combined with the high totipotency of many dicot leaf cells and developed into a simple leaf-disc transformation system in which gene transfer, selection and regeneration are coupled together in an efficient process (50). Tobacco is an excellent host for Agrobacterium. It responds very well in in vitro culture and was the first plant species transformed using Agrobacterium. Advances in tissue-culture regeneration techniques and effective interaction with A. tumefaciens have provided a reproducible method for transfer of cloned, engineered genes in many dicot crop species including tomato, lettuce, sunflower, rape, cotton, and soybean (51). Recently, Agrobacterium-mediated transformation of so-called non-host crop plants has been accomplished in maize and rice (49, 52).
There are a number of technical issues that have required attention in the application of this technique to species other than model species (e.g. tobacco). One problem is inefficiency or ineffectiveness of the selectable marker. Escapes or false positives often occur, perhaps due to loss of DNA during plant development or due to the cross-protection of wild-type cells by nearby transformed cells. This problem generally has been solved by applying selection during more susceptible growth stages or using dual selection. Some plant species or genotypes respond differently to the same selection agent even though they are transformed with the same marker gene. Therefore, new marker genes and selection agents have been developed to use in those crops. Another problem has been the interaction between Agrobacterium and the explant, sometimes limiting the survival or vigor of the explants which, in turn, reduces the overall transformation efficiency. The strain of Agrobacterium can have significant impact on the efficiency of transformation (71).
Cellular competency for transformation and regeneration remains a major barrier for application of this procedure. One example is the sugar beet (Beta vulgaris L.). Early attempts to transform sugar beet based on Agrobacterium-mediated gene transfer demonstrated that cells are readily susceptible to transformation. However, the transgenic calli obtained were non-regenerable (62) or the recovery frequency of transgenic plants was very low (33). Recently, Hall et al. (40) reported a high-efficiency transgenic sugar beet system using stomatal guard cell protoplasts. He used computer-assisted microscopy that identified guard cells as the only progenitor or totipotent protoplasts in sugar beet leaves. This regenerable guard cell line could be a good candidate for Agrobacterium-mediated transformation. However, a non-uniform regeneration response was observed from initial cultures derived from guard cells, making this system unpredictable.
Particle Bombardment
Microprojectile bombardment, a transformation procedure, employs high velocity metal particles coated with DNA to deliver genes directly into cells. It can apply to a wide spectrum of experimental systems including plants, insects, fish and mammals (113). In plants, the concept has been described by Sanford (89) following the original observation by Klein et al. (59) that tungsten particles could be used to introduce macromolecules such as DNA and RNA into epidermal cells of onion with subsequent transient expression of the enzymes encoded by the DNA. Christou et al. (18) then demonstrated that the process could be used to deliver foreign genes into plant cells and stably integrate them into the genome. Particle bombardment is a direct method which can target any organ or tissue with single or multiple genes. Theoretically, it can be a genotype-independent transformation procedure if the targeted plant cells are totipotent. It limits or eliminates the time that the transformed cell line remains in tissue culture and thus reduces the risk of losing fertility due to tissue-culture-induced somaclonal variation. An efficient particle bombardment system for crops after the optimization of physical parameters requires target cells that are competent for both stable DNA integration and regeneration. Many crop species that are poor hosts for Agrobacterium and not efficiently transformed through protoplast-based procedures are candidates for particle bombardment. Fertile transgenic plants have been successfully obtained from crops that cannot be achieved by any other method, including wheat (108, 109). However, transformation frequency remains extremely low and only a few genotypes can be manipulated.
Viral Vectors for Gene Expression
The development of plant viruses as vectors to deliver foreign genes into plant cells has become an attractive tool to complement the currently available methodology (91), although it too has significant limitations. The decision to employ plant virus vectors over traditional plant breeding or transformation depends upon the goals of the application, as well as upon technical feasibility.
The potential advantages of using viral vectors include speed, flexibility of application, high levels of gene expression, and non-transmissibility through seed generations. Numerous plants of the same genotype or different genotypes of the same species can be infected with a virus to evaluate expression of a foreign gene. In addition, viruses carrying different genes of interest can be used singly or in combination, applied at the needed time to combat a specific problem or set of problems. Because of the nature of virus infection, the maximum level of foreign gene expression is predicted to occur within a brief period, usually within one or two weeks after inoculation. Another practical advantage is that many plant viruses are easily transmissible and potentially could be used for rapid mechanical inoculation of large acreages of crop plants. Also, once a suitable transient gene vector is identified, the expression of large numbers of genes can be tested rapidly in a variety of different plants that are hosts for the virus. As most viruses are not transmitted through the host germ line, the potential for environmental escape of foreign genes is limited.
Both DNA and RNA viruses have the potential to be effective vectors for gene delivery. It was generally believed that all plant viruses were RNA viruses until Shepherd (95) reported the cauliflower mosaic virus (CaMV) contains a double-stranded DNA genome. Cloned CaMV DNA was observed to be infectious when plants were mechanically inoculated (95, 96). This report resulted in an explosion of research on this and other plant virus systems. The CaMV 35S promoter has been used extensively as a promoter for constitutive expression of foreign genes. Today, the reverse transcription of viral RNA into cDNA that can be inserted into plasmid vectors for molecular cloning (3) and the in vitro transcription systems that facilitate the synthesis of infectious RNA transcripts from full-length cDNA clones (12) make feasible the use of RNA viruses as potential vectors.
Many strategies have been used to develop virus-based gene-delivery vectors. The main criterion for effectiveness of viral gene manipulation has been lack of disruption of essential viral functions. Strategies include gene replacement, gene insertion, complementation and artificial virions (91, 114).
Gene replacement. To reduce the possible detrimental effects of increased genome size when introducing a foreign gene into the viral genome, it has been proposed that viral genes be replaced by foreign genes. Many viruses have been tested as vectors for foreign gene expression using gene replacement, including CaMV, tobacco mosaic virus (TMV), potato virus X (PVX), tomato golden mosaic virus (TGMV), and African cassava mosaic virus (ACMV). For CaMV, mainly small genes have been replaced and expressed. The molecular recombination caused by rapid deletion of foreign inserts has limited its gene-vector potential (13). TMV, a single-stranded, plus-sense, rod-shaped RNA virus, theoretically can overcome paclaging constraints imposed by spherical virus coat proteins and could be more flexible and stable for expression of a foreign gene. When chloramphenicol acetyltransferase (CAT) replaced the TMV coat protein, high levels of expression failed to spread efficiently throughout the plant, because the coat protein is required for efficient long distance movement (21). This also occurred with PVX when the .beta.-glucuronidase (GUS) gene was substituted (16). Replacement of the coat protein gene of TGMV and ACMV, both single-stranded DNA geminiviruses, resulted in vectors replicated and spread throughout infected plants, with concomitant high levels of foreign gene expression. Even though the DNA was not encapsidated, it appears that unknown structural features dictated that vectors were stable when their size was comparable to that of the wild type genome (44, 101).
Gene insertion. Gene replacement may affect the expression of the normal viral gene. To avoid such potentially negative effects, gene insertion or addition has been tested using several viruses, including TMV, maize streak virus (MSV), PVX, and tobacco etch virus (TEV). Duplication of a homologous coat protein subgenomic promoter has been shown to be a useful approach for the expression of foreign genes from PVX in whole plants. Insertion of the GUS gene downstream of the duplicated promoter sequence resulted in stable, strong GUS expression throughout the plants. However, GUS deletions did occur over time (16). Using TMV, this approach resulted in homologous recombination and loss of the foreign insert (63). However reducing the sequence's relatedness decreased the frequency of recombination events between the two subgenomic promoters, thus providing a more stable vector (28). For MSV, a geminivirus, systemic infection requires all of the intact viral genes without interruption (93), although localized expression of a foreign gene was obtained when it was inserted in the intercistronic region. The expression strategy of potyviruses like TEV involves cis-regulated proteolytic polyprotein processing steps, and as a consequence, foreign genes could not be simply inserted between other viral genes. These genes could be expressed either through insertional fusion with existing genes or through inclusion of appropriate adjacent proteolytic cleavage sites that permitted efficient protein processing and release of the foreign gene product along with the essential viral products (27). Although deletion events did eventually occur, the TEV vector was sufficiently stable to maintain GUS gene expression for several mechanical passages through plants.
Complementation systems. In the examples mentioned above, foreign genes are expressed from autonomously replicating viruses that have the potential to invade the plant systematically. Although these strategies have obvious advantages, limitations are imposed by the fact that most replacements of viral genes affect essential function, whereas gene insertion increases the genome size which may impact the ability to be packaged. To overcome some of these problems, helper-dependent systems have been explored in which foreign genes are inserted into defective viral components that either depend on transgenically introduced viral genes or on co-infection with a helper virus for essential function. The potential of this strategy has not yet been fully explored, but the feasibility of transgenic complementation of viral function has been demonstrated for a number of virus systems (25, 35, 107). This complementation approach could possibly be exploited for gene replacement strategies.
Artificial virions. A completely different approach to using plant viruses and their natural vectors for delivery of foreign genes to plants is that described by Zhang et al. (114). Opidium brassicae is a primitive zoosporic fungus that vectors tobacco necrosis virus (TNV) and several other viruses to numerous monocot and dicot species. A recombinant plasmid containing the CAT gene was encapsidated with the dissociated coat protein of TNV. Zoospores of O. brassicae were able to acquire these "pseudovirions" and transmit them to wheat roots. Two days after inoculation, expression of CAT could be detected in infected roots. The fate of the encapsidated plasmid in infected cells was not discussed. The authors indicated that this system might "provide an alternative" to "agro-infection" and particle-gun systems.
Limitations of Current Gene Delivery Systems
All of the above processes are highly technical, expensive, lengthy and difficult to control. The situation is often complicated by the incompetency or special requirements for competency of specific plant genotypes. Other important difficulties related to available transformation methods are poor expression of transgenes, loss or inactivation of transgenes and low regeneration frequency in some species or genotypes. Expression may be hampered by position effect or problems involving copy number (19, 20). Expression may be optimized through selection and screening of numerous independent transformants, though this is laborious and time consuming (100). Loss or inactivation of transgenes is frequently related to tissue-culture-generated somaclonal variation (65). Significant progress has been made in developing improved regeneration frequency, though this has occurred through empirical approaches that continue to defy a general understanding of genotypic variation in regeneration frequency (65). In addition to these difficulties, once seed containing a foreign gene is released, the gene can be transmitted to subsequent generations, so that control of proprietary genes requires substantial resources. In addition, effective use of transgenic crops may be limited since many crops can cross pollinate with their weedy relatives. Thus, the potential exists for transfer of foreign genes into these weed species, with undesirable environmental consequences.
The use of plant viruses as vectors of foreign genes has numerous advantages over more traditional methods of introducing genes into plants; however, there are certain problems associated with the use of plant virus vectors that could potentially minimize their usefulness. There are technical difficulties related to packaging, stability and expression, and agricultural-application problems related to containment and host specificity of the virus vector and associated foreign genes to the targeted host species.
Rapid recombination of the modified virus appears to be the predominant cause of instability and deletion of foreign genes (99). Instability problems are more prevalent when large genes are inserted rather than small genes (91). The mechanism responsible for deletion is unclear, however, the ability of viruses to delete foreign inserts may reflect a universal strategy that has evolved to protect viruses against promiscuous incorporation of host genes and subsequent amplification of non-advantageous sequences. Since many unknown and currently unpredictable factors may contribute to instability, it is important to test the effects of a variety of gene insertion sites and strategies in exploring the use of any virus as the vector of foreign genes. Other technical limitations to the use of viral-based vectors include the usually brief period of maximum expression, and poor understanding of host delivery and movement mechanisms.
By definition, plant viruses are pathogens and are capable of causing disease and reducing yields in infected plants. Rhizomania, "root madness" is a severe disease of sugar beet caused by beet necrotic yellow vein virus (BNYVV) and transmitted by the soilborne fungus Poymyxa betae. The disease is widely distributed in many countries and is economically devastating to a sugar beet crop, causing severe loss in root yield and sugar content of the infected plants. In naturally infected plants, BNYVV is normally confined to the roots, and causes massive proliferation of the lateral rootlets of taproots as well as other abnormalities of the root system.
Although some virus vectors have been genetically modified to reduce the virulence of plant viruses (91) most have not. Furthermore, many of the viruses used as vectors have extremely wide host ranges and infect both monocots and dicots (91). Even if a naturally mild virus isolate is intended for use as a vector with a specific host, it could potentially be virulent to a non-target species. Fear that an introduced virus might move from the intended target plant to a non-target host is one reason cross protection has not been widely adopted as a means of biological control of plant virus diseases (34, 86, 96). The possibility that a recombinant virus could escape into the environment and introduce a foreign gene into a non-target species constitutes an even greater constraint to the development and use of virus-vector-delivery systems in production agriculture. Viruses that have large host ranges and are easily transmitted mechanically or by insects can be subjected to extreme scrutiny from regulatory agencies and the public. Even though there are certain advantages to selecting viruses with large host ranges as virus vectors, regulatory concerns may dictate the development of systems with vectors that are more host specific. Such a mandate would be in line with the shift in pesticide use that has occurred over the last ten years, from broad spectrum pesticides to those with more specific modes of action.
Novel isolates of beet soilborne mosaic virus, BSBMV (previously called Texas 7), have now been identified as non-virulent or mildly virulent viruses closely related to BNYVV. When co-inoculated with BNYV in a susceptible host, BSBMV dominates over BNYVV, and interferes with infection by the more pathogenic BNYVV. A method for preferentially incorporating BSBMV into plants and thereby excluding or inhibiting infection and disease caused by the more virulent BNYVV would be extremely useful, especially in the sugar beet industry. Such a method would also be of great utility for inoculation of plants against a variety of severe diseases caused by furoviruses.
Similarly, a method for easily incorporating a competing, non-virulent or mildly virulent virus into a plant to confer protection against a more virulent pathogen would be highly desirable. Such a method should be easily applied in the field, permit specific application to a plant or field of choice, and be stable over time. The methods of the present invention provide such easily administered and controlled application of a beneficial virus or other desired nucleic acid sequence to a host plant.
In addition, the methods of the present invention describe a novel gene delivery system that uses the natural fungal vector and a seed coating treatment. The invention method results in products specific to the crop of interest and the trait (or set of traits) of interest. It can be applied quickly and easily by the grower without the need for specific training. Further, since the gene(s) is (are) not incorporated into the plant's genome, and is therefore not genetically transmissible, the manufacturer is in an excellent position to control the release and distribution of the product(s). Owing to the obligate nature of the relationship among host plant, fungal vector and virus, opportunities for unintended transmission of gene is minimal.
In addition, the methods of the present invention relate to the delivery of recombinant or wildtype viruses to cells via the infection of plants by soilborne fungi containing wildtype or recombinant viruses.
The development of a seed treatment system for viral delivery as well as virus-mediated gene delivery to plants is a novel concept which fits well with the goal of achieving sustainability of U.S. agriculture. It will improve productivity by increasing crop quality, and value, which, in turn, will improve the economic viability of farm operations. Also, such a system could potentially enhance environmental quality by, for example introducing resistance genes into plants which could reduce requirements to pesticide use.